Bottom Line:
Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles.Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%.These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

ABSTRACTIn the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

fig5: Physical association of actin with intracytoplasmic vacuoles. (a) F-actin is present in filopodia and adhesion plaques in the trophozoite; at 12 h, F-actin is surrounding structures with vacuolar appearance (arrows); at 24 and 48 h, F-actin was concentrated in a region of the amoeba (arrows); at 72 h, F-actin is located in structures with vesicular appearance (arrows); at 96 h, F-actin localized in the periphery of the cyst (arrows). F-actin was detected by Rhodamine-phalloidin (1 : 50) by confocal microscopy. Images are representative of two independent experiments. (b) Ultrastructure of the encystment process. The elimination of vacuoles, surrounded by a cytoplasm rich in thin filaments (arrows), characteristic appearance of high actin content, was observed at early times after induction of encystment (12 h). Samples were processed as described in Section 2.

Mentions:
To analyze the type and location of structures formed by F-actin during encystment, samples were analyzed by confocal microscopy. Trophozoites showed actin filaments abundantly in the periphery of the cell, in filopodia, in adhesion plaques, and in phagocytic structures (Figure 5(a), T). At 12 h after induction of encystment, F-actin appeared surrounding vacuoles (arrows), whereas at 24 h the number of these structures was reduced, at 48 h mostly a single but larger cluster of actin was observed in the center of the amoebas. At 72 h smaller structures of F-actin with vesicular appearance were seen, and at 96 h scarce dots of F-actin were found decorating the periphery of the cysts (Figure 5(a), 12–96 h).

fig5: Physical association of actin with intracytoplasmic vacuoles. (a) F-actin is present in filopodia and adhesion plaques in the trophozoite; at 12 h, F-actin is surrounding structures with vacuolar appearance (arrows); at 24 and 48 h, F-actin was concentrated in a region of the amoeba (arrows); at 72 h, F-actin is located in structures with vesicular appearance (arrows); at 96 h, F-actin localized in the periphery of the cyst (arrows). F-actin was detected by Rhodamine-phalloidin (1 : 50) by confocal microscopy. Images are representative of two independent experiments. (b) Ultrastructure of the encystment process. The elimination of vacuoles, surrounded by a cytoplasm rich in thin filaments (arrows), characteristic appearance of high actin content, was observed at early times after induction of encystment (12 h). Samples were processed as described in Section 2.

Mentions:
To analyze the type and location of structures formed by F-actin during encystment, samples were analyzed by confocal microscopy. Trophozoites showed actin filaments abundantly in the periphery of the cell, in filopodia, in adhesion plaques, and in phagocytic structures (Figure 5(a), T). At 12 h after induction of encystment, F-actin appeared surrounding vacuoles (arrows), whereas at 24 h the number of these structures was reduced, at 48 h mostly a single but larger cluster of actin was observed in the center of the amoebas. At 72 h smaller structures of F-actin with vesicular appearance were seen, and at 96 h scarce dots of F-actin were found decorating the periphery of the cysts (Figure 5(a), 12–96 h).

Bottom Line:
Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles.Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%.These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

ABSTRACTIn the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.